1. Field of the Invention
This invention relates to a method of enhancing the activity of lysosomal xcex1-Galactosidase A (xcex1-Gal A) in mammalian cells and for treatment of glycosphingolipid storage diseases, in particular Fabry disease, by administration of 1-deoxy-galactonojirimycin and related compounds.
2. Background Information
Fabry disease (1) is a glycosphingolipid lysosomal storage disease caused by an X-linked inherited deficiency of lysosomal xcex1-galactosidase A (xcex1-Gal A), an enzyme responsible for the hydrolysis of terminal xcex1-galactosyl residue from glycosphingolipids. A deficiency in the enzyme activity results in a progressive deposition of neutral glycosphingolipids, predominantly globotriaosylceramide (ceramide trihexoside, CTH), in vascular endothelial cells causing renal failure along with premature myocardial infarction and strokes in patients with this condition (2). This disorder is classified by clinical manifestations into two groups: a classic form with generalized vasculopathy and an atypical variant form, with clinical manifestations limited to heart. Recently, the atypical variant of the disease was found in 10% of adult male patients with unexplained left ventricular hypertrophy, increasing the estimation of frequency for the disorder (3). Like other glycosphingolipid lysosomal storage diseases, enzyme replacement therapy, gene therapy, bone marrow transplantation, and substrate deprivation are suggested as potential strategies for the treatment of this disease (4). However, at the moment the only treatment for this disorder is symptomatic management. Therefore, development of a new therapeutic strategy for this disease is urgently needed.
Studies (5) on residual xcex1-Gal A activity of mutant enzymes revealed that some of mutant enzymes have similar kinetic properties to normal xcex1-Gal A but with significant instability. This is considered as the case for most of atypical variant patients who generally showed higher residual xcex1-Gal A activity than classical Fabry patients. For example (6), a purified mutant xcex1-Gal A with a genotype of Q279E, found in a patient with atypical variant of Fabry disease, had the same Km and Vmax as the normal enzyme, but lost most of the enzyme activity by incubating the enzyme at pH 7.0 at 37xc2x0 C. for 30 min while the normal enzyme was stable under the same condition. Both mutant and normal enzymes were stable at pH 5.0 at 37xc2x0 C. Furthermore, the majority of the mutant enzyme protein in cells formed aggregate in endoplasmic reticulum (ER) and was quickly degraded (7), suggesting that the deficiency of the enzyme activity in this mutant maybe primarily caused by the unsuccessful exit of ER leading to excessive degradation of the enzyme protein. The present invention focuses on the aid of smooth escape of the enzyme from ER to prevent the degradation of the mutant enzyme.
The strategy of the invention is based on the following model. The mutant enzyme protein tends to fold in an incorrect conformation in ER where the pH is around 7. As a result, the enzyme is retarded from the normal transport pathway from ER through the Golgi apparatus and endosome to the lysosome, but instead is subjected to degradation. On the other hand, the enzyme protein with a proper conformation is transported to the lysosome smoothly and remains in an active form because the enzyme is more stable at a pH of less than 5. Therefore, a compound which is able to induce a proper conformation in mutant enzyme may serve as an enhancer for the enzyme. The present inventors have unexpectedly found that strong competitive inhibitors for xcex1-Gal A at low concentrations enhance the mutant enzyme activity in cells, including mutant xcex1-Gal A gene transfected COS-1 cells, fibroblasts from a transgenic mouse overexpressing mutant xcex1-Gal A, and lymphoblasts from Fabry patients.
It is noted that while the above is believed to be the mechanism of operation of the present invention, the success of the invention is not dependent upon this being the correct mechanism.
Accordingly, it is one object of the present invention to provide a method of preventing degradation of mutant xcex1-Gal A in mammalian cells, particularly in human cells.
It is a further object of the invention to provide a method of enhancing xcex1-Gal A activity in mammalian cells, particularly in human cells. The methods of the present invention enhance the activity of both normal and mutant xcex1-Gal A, particularly of mutant xcex1-Gal A which is present in certain forms of Fabry disease.
In addition, the methods of the invention are also expected to be useful in nonmammalian cells, such as, for example, cultured insect cells and CHO cells which are used for production of xcex1-Gal A for enzyme replacement therapy.
Compounds expected to be effective in the methods of the invention are galactose and glucose derivatives having a nitrogen replacing the oxygen in the ring, preferably galactose derivatives such as 1-deoxygalactonojirimycin and 3,4-diepi-xcex1-homonojirimycin. By galactose derivative is intended to mean that the hydroxyl group at the C-3 position is equatorial and the hydroxyl group at the C-4 position is axial, as represented, for example, by the following structures: 
wherein R1 represents H, methyl or ethyl; R2 and R3 independently represent H, OH, a simple sugar (e.g. xe2x80x94Oxe2x80x94galactose), a 1-3 carbon alkyl, alkoxy or hydroxyalkyl group (e.g. CH2OH).
Other specific competitive inhibitors for (x-galactosidase, such as for example, calystegine A3, B2 and B3, and N-methyl derivatives of these compounds should also be useful in the methods of the invention. The calystegine compounds can be represented by the formula 
wherein for calystegine A3: R1=H, R2=OH, R3=H, R4=H; for calystegine B2: R1=H, R2=OH, R3=H, R4=OH; for calystegine B3: R1=H, R2=H, R3=OH, R4=OH; for N-methyl-calystegine A3: R1=CH3, R2=OH, R3=H, R4=H; for N-methyl-calystegine B2: R1=CH3, R2=OH, R3=H, R4=OH; and for N-methyl-calystegine B3: R1=CH3, R2=H, R3=OH, R4=OH.
It is yet a further object of the invention to provide a method of treatment for patients with Fabry disease. Administration of a pharmaceutically effective amount of a compound of formula 
wherein
R1 represents H, CH3 or CH3CH2;
R2 and R3 independently represent H, OH, a 1-6 carbon alkyl, hydroxyalkyl or alkoxy group (preferably 1-3), or a simple sugar;
R4 and R5 independently represent H or OH; or a compound selected from the group consisting of 2,5-dideoxy-2,5-imino-D-mannitol, xcex1-homonojirimycin, 3,4-diepi-xcex1-homonojirimycin, 5xe2x80x94Oxe2x80x94xcex1-D-galactopyranosyl-xcex1-homonojirimycin, 1-deoxygalactonojirimycin, 4-epi-fagomine, and 1-Deoxy-nojirimycin and their N-alkyl derivatives, will alleviate the symptoms of Fabry disease by increasing the activity of mutant xcex1-Gal A in patients suffering from Fabry disease. Other competitive inhibitors of xcex1-Gal A, such as calystegine compounds and derivatives thereof should also be useful for treating Fabry disease.
Persons of skill in the art will understand that an effective amount of the compounds used in the methods of the invention can be determined by routine experimentation, but is expected to be an amount resulting in serum levels between 0.01 and 100 xcexcM, preferably between 0.01 and 10 xcexcM, most preferably between 0.05 and 1 xcexcM. The effective dose of the compounds is expected to be between 0.5 and 1000 mg/kg body weight per day, preferably between 0.5 and 100, most preferably between 1 and 50 mg/kg body weight per day. The compounds can be administered alone or optionally along with pharmaceutically acceptable carriers and excipients, in preformulated dosages. The administration of an effective amount of the compound will result in an increase in xcex1-Gal A activity of the cells of a patient sufficient to improve the symptoms of the patient. It is expected that an enzyme activity level of 30% of normal could significatly improve the symptoms in Fabry patients, because the low range of enzyme activity found in apparently normal persons is about 30% of the average value (2).
Compounds disclosed herein and other competitive inhibitors for xcex1-Gal A which will be known to those of skill in the art will be useful according to the invention in methods of enhancing the intracellular activity of xcex1-Gal A and treating Fabry disease.